LASIK is a surgical procedure involving a two-step process: a femtosecond laser is first used to cut a flap in the cornea with the subsequent use of an excimer laser for corneal surface ablation.1,2 As an alternative to the current LASIK procedure, femtosecond lenticule extraction eliminates the excimer laser and performs refractive surgery solely with a femtosecond laser. The earliest version of this technique used an Nd:YLF picosecond laser to create a traditional LASIK flap and cut a refractive lenticule in the stroma.3,4 The femtosecond laser was later substituted for the picosecond laser (femtosecond lenticule extraction).5–10 The procedure was later improved by creating an intrastromal pocket instead of a LASIK flap (SMILE); it is created using the femtosecond laser with a diameter similar to that of a LASIK flap, with a 3- to 5-mm opening at the hinge position.11–14 The refractive lenticule is removed through this smaller opening in the cornea. In both versions of the technique, the exact geometry and thickness of the refractive lenticule is calculated based on the refractive error of the patient.6
Early results from the femtosecond lenticule extraction and SMILE trials were promising with a high rate of patient satisfaction.5–13,15,16 However, preliminary investigations of the surface quality of lenticules produced using the 200-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Dublin, CA) with pulse energies ranging from 150 to 195 nJ showed evidence of circular holes from cavitation bubbles and rough patches.7,17,18 A later study looked at the surface quality of the stromal bed after lenticule extraction.7 Although a next generation VisuMax laser was used with a higher repetition rate (500 kHz) and lower pulse energy (130 nJ), circular holes and rough areas were still visible in scanning electron microscopy images. Corneal surface quality is vital to predicting postoperative patient satisfaction because a smooth stromal surface may be associated with better optical quality, a diminished inflammatory response during healing, and faster visual recovery.18–23 Therefore, the current study was designed to investigate the surface smoothness of refractive lenticules removed during SMILE using the 500-kHz Visumax femtosecond laser with a low pulse energy and smaller spot separation than ones used in previous studies.
Patients and Methods
Intrastromal lenticules were retrieved from 8 eyes of 5 patients who underwent SMILE at the Aarhus University Hospital in Denmark. The samples were obtained and used in compliance with the guidelines of the Declaration of Helsinki for research involving the use of human tissue, and imaging experiments were approved by the University of Miami’s Institutional Review Board. Only patients who required myopia correction (no cylinder) were included in this study.
The VisuMax femtosecond laser system was used to perform SMILE. The same surgeon (JØH) performed the procedure for all samples included in this study. Femtosecond laser pulses (500 kHz, cut energy index 26, equivalent to an energy of 130 nJ) were focused in a spiral pattern with a 2.5 × 2.5 μm spot/track separation. Cut surfaces were created beginning with the most posterior surface, moving toward successively anterior surfaces, and finishing with a 60° incision located at the 12-o’clock position (Figure 1). The lenticule diameter (optical zone) was 6.5 mm with a minimum edge thickness of 15 μm and the cap diameter was 7.3 mm with an intended thickness of 120 μm. After laser treatment, the lenticule was first freed from any remaining tissue bridges by manually separating the anterior and posterior dissection planes using a spatula and then extracted with forceps.
(A) Side view of the small incision lenticule extraction cap (downward diagonal shading) and refractive lenticule (upward diagonal shading) relative to full corneal thickness. (B) Side view of lenticule extraction and relaxation of the cap to corrected curvature. (C) Top view of the cap and lenticule. The bolded area illustrates the location of the pocket opening through which the lenticule was removed. (D) Top view of lenticule extraction through the pocket opening.
Scanning Electron Microscopy
The extracted lenticules were placed in 2% formalin and shipped to the University of Miami (Coral Gables, FL) for imaging with an environmental scanning electron microscope (eSEM). The eSEM is an adaptation of the traditional SEM that enables imaging of hydrated, uncoated biological samples. A series of pressure-limiting apertures maintains a pressure differential down the electron beam column so that the electron source is under an ultra-high vacuum, whereas the sample is under a low vacuum. An electron beam is scanned across the sample, which in turn ejects a signal in the form of secondary electrons. These secondary electrons are amplified by the presence of water vapor from the hydrated sample during a process known as gas cascade amplification. A gaseous detector device is used to collect the signal generated by the sample to create an image. The use of eSEM to image biological samples eliminates artifacts that occur due to sample dehydration and coating required for traditional SEM imaging.24
Surface Quality Analysis
Surface quality of intrastromal lenticules was evaluated by one investigator (NMZ) who specializes in electron microscopy. The smoothness of both the anterior and posterior surfaces of the lenticules was assessed from images obtained at three specific magnifications (100×, 250×, and 500×) in the central zone. A roughness scale based on one used previously by Wilhelm et al.25 to evaluate corneal surface quality after sectioning with different microkeratomes was applied to our analysis. Three criteria were used to grade surface roughness: overall surface regularity, percent of surface irregularity, and position of irregular area. The focus of the analysis was determining if specific features associated with intrinsic roughness were present in the images. These features generally resembled small-scale tears or attachments of collagen fibers, often appearing as high-contrast lines or networks. The presence of holes that could be due to cavitation bubbles was also noted. Other features (eg, waviness or particulate contamination) were ignored.
Eight intrastromal lenticules created using the Visumax femtosecond laser were included in this study. Mean lenticule diameter was 6.46 ± 0.07 mm (range: 6.3 to 6.5 mm). Mean preoperative central corneal thickness was 537 ± 18 μm (range: 502 to 565 μm). Mean residual bed thickness was 283 ± 21 μm (range: 256 to 328 μm). Mean intended lenticule central thickness was 134 ± 12 μm (range: 117 to 152 μm) (Table 1, Figure 2).
Myopia Correction, Corneal Thickness, and Lenticule Dimensions of 8 Eyes
Illustration of corneal lenticule geometry. The minimum edge thickness was 15 μm and the intended central thickness ranged from 117 to 152 μm depending on the spherical correction required.
Example images obtained of the corneal lenticules are shown in Figures 3–4. The results of the roughness analysis for all samples (top and bottom) are given in Table 2. Of the eight samples, three showed surface irregularities on one side only (sample 1 bottom and samples 6 and 8 top). Overall, both surfaces of the lenticule appeared extremely smooth and absent of significant surface irregularities. The smoothness appeared the same for the top and bottom surfaces. In addition, the smoothness appeared the same for the range of correction included in this study (−6.75 to −10 diopters). No holes from cavitation bubbles were seen in any of the images. Edges were mostly smooth, but jagged in areas where the surgeon removed the lenticule with forceps (Figure 5).
Environmental scanning electron microscope images of the top surface of the corneal lenticules taken at 100× (top), 250× (middle), and 500× (bottom). Low magnification images contain the microscope field aperture. The left column corresponds to sample 4 and the right column corresponds to sample 8. D = diopters
Environmental scanning electron microscope images of the bottom surface of the corneal lenticules taken at 100× (top), 250× (middle), and 500× (bottom). Low magnification images contain the microscope field aperture. The left column corresponds to sample 4 and the right column corresponds to sample 8. D = diopters
Summary of Surface Quality Results for Each Sample
Folded lenticule sample, illustrating the regularity of the edge (left). Lenticule edge with clear marks from the forceps used to remove the lenticule (right). Original magnification 100× and low magnification images contain the microscope field aperture.
Surface quality of the stroma is vital to optimal vision postoperatively because previous studies have demonstrated a direct correlation between surface quality and postoperative haze and refraction.18–23 Therefore, several studies investigated the smoothness of the corneal lenticule surface produced by the new femtosecond lenticule extraction and SMILE. The first studies were performed in an ex vivo pig model using the VisuMax laser with a 200-kHz repetition rate, 185-nJ pulse energy, and 3 × 3 μm spot separation.17 Lenticules of predictable surface quality were produced, although the authors noted that the quality was somewhat lower than that of lenticules cut with a mechanical microkeratome and reported some surface irregularities such as roughness due to tissue bridges, cavitation bubbles, and grooves. Kunert et al.18 published an analysis of the quality of lenticules extracted from patients using a 200-kHz VisuMax system with a 3 × 3 μm spot separation. The authors used three different pulse energies (150, 180, and 195 nJ) to study how this parameter affects lenticule cut quality. The stromal bed, lenticule appearance, and ease of dissection were subjectively graded on the basis of an established scale.25 The best quality resections were generated with the lowest pulse energy used in the study (150 nJ), suggesting that lower energy levels produced smoother surfaces. Most recently, the new generation VisuMax femtosecond laser with a 500-kHz repetition rate, 130-nJ pulse energy, and 3 × 3 μm spot separation was used to create refractive lenticules in cadaver eyes.7 Although the surface quality improved from the earlier studies, spots assumed to be from cavitation bubbles and rough patches were still clearly visible.
Based on these previous studies, it can be assumed that pulse energy and laser frequency are extremely important variables for optimizing surface quality when cutting the cornea with a femtosecond laser. During photodisruption of the stroma, small cavitation bubbles formed at the interface. Bubble occurrence can be reduced by decreasing the pulse energy and increasing the laser frequency, enabling a more homogeneous cut.26 In the current study, we used the 500-kHz VisuMax laser and a low pulse energy. The experimental parameters used in the current study may explain why the surfaces of all samples imaged (top and bottom) were smooth and absent of significant surface irregularities and holes from cavitation bubbles.
Another important parameter in laser-assisted refractive surgery is spot separation. Because the femtosecond laser works through photodisruption of the stroma at its focal plane, many collagen fibers remain attached after sectioning.27 Initial studies investigating the morphology of LASIK flaps found that a smaller spot separation caused less trauma to the flap and underlying stroma when the flap was lifted.28 In previous investigations of lenticule surface quality, a 3 × 3 μm spot separation was used.7,17,18 In the current study, the spot separation was reduced to 2.5 × 2.5 μm. Closer spot placement likely leaves fewer collagen fibers intact, which would allow for more efficient and easier lenticule extraction. Reducing laser spot separation could be an important method of reducing surface roughness.
Because all previous studies used SEM to image lenticule samples,7,17,18 some of the roughness observed could also be due to the significant preparation (critical point drying and gold sputter coating) required before imaging that can modify the ultrastructure of delicate tissues. During the dehydration process, samples can shrink up to 60% and this contraction can often result in cracking.24 In the current study, eSEM was used to image various lenticule surfaces. The eSEM is an adaptation of the traditional SEM that enables imaging of hydrated, uncoated biological samples, thereby eliminating artifacts due solely to tissue preparation.24
Immediately after removal from the eye, the lenticules were placed in a light fixative (2% formalin). No additional preparation was required prior to imaging the samples under low vacuum conditions. Because of this, the images acquired were free of artifacts due solely to sample preparation. However, because the samples were still malleable, it was difficult to place them completely flat on the imaging surface. This resulted in appearances of some folds and wrinkles in the images. Although the presence of water on the surface of the samples during eSEM imaging protects the sample from dehydration, it may reduce contrast and obscure smaller surface details.24,29 In addition, the release of vapor during imaging may actually cause the overall image to appear out of focus. Therefore, eSEM and SEM are typically used in tandem to obtain more complete information about the sample’s structure. In the current study, all samples were prepared and imaged with SEM after eSEM. Although the samples had undergone significant shrinkage, no surface irregularities described in previous studies were observed.
Although eSEM is a powerful technique for imaging delicate biological samples, it is important to note that a lower magnification and a shorter amount of time for imaging must be used to minimize damage to the sample.29 Two of the samples imaged in the current study had wispy fibers running across that were most likely caused by mild heating of the surface by the electron beam during extended imaging with the eSEM.
The new generation VisuMax laser operating at 500 kHz with a pulse energy of 130 nJ and a spot/track separation of 2.5 × 2.5 μm was able to produce smooth refractive lenticules. Further prospective studies are required to confirm these encouraging results and to assess whether lenticule surface quality after astigmatic correction is as smooth as after myopic correction.
- Solomon KD, Fernandez de Castro LE, Sandoval HP, et al. LASIK world literature review: quality of life and patient satisfaction. Ophthalmology. 2009;116:691–701. doi:10.1016/j.ophtha.2008.12.037 [CrossRef]
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- Krueger RR, Marchi V, Gualano A, Juhasz T, Speaker M, Suarez C. Clinical analysis of the neodymium: YLF picosecond laser as a microkeratome for laser in situ keratomileusis: partially sighted eye study. J Cataract Refract Surg. 1998;24:1434–1440. doi:10.1016/S0886-3350(98)80163-6 [CrossRef]
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- Sekundo W, Kunert K, Russmann C, et al. First efficacy and safety study of femtosecond lenticule extraction for the correction of myopia: six-month results. J Cataract Refract Surg. 2008;34:1513–1520. doi:10.1016/j.jcrs.2008.05.033 [CrossRef]
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- Kamiya K, Igarashi A, Ishii R, Sato N, Nishimoto H, Shimizu K. Early clinical outcomes, including efficacy and endothelial cell loss, of refractive lenticule extraction using a 500 kHz femtosecond laser to correct myopia. J Cataract Refract Surg. 2012;38:1996–2002. doi:10.1016/j.jcrs.2012.06.052 [CrossRef]
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Myopia Correction, Corneal Thickness, and Lenticule Dimensions of 8 Eyesa
|Sample||Spherical Equivalent (D)||Central Corneal Thickness (μm)||Residual Bed Thickness (μm)||Lenticule Diameter (mm)||Intended Lenticule Central Thickness (μm)|
Summary of Surface Quality Results for Each Sample
|Sample||Evaluation of Surface Irregularity|
|Bottom||Some roughness||25% coverage||Contained to top of field of view|
|6||Top||Some roughness||25% coverage||All over|
|8||Top||Some roughness||< 10% coverage||Contained along diameter of sample|